Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder that causes excessive proliferation and rapid accumulation of myeloid precursor cells in the bone marrow. If left untreated, death occurs within weeks or months after diagnosis. AML is a heterogeneous disease. In the sub-group of promyelocytic leukemia (PML) with a specific chromosome translocation fusing the genes for the PML and RAR proteins, retinoic acid based differentiation therapy, in combination with an anthracycline drug, and sometimes the differentiation enhancer arsenic trioxide, has proven successful (P. Fenaux, C. Chastang, S. Chevret et al., Blood 94 (4), 1192 (1999). Improved therapy has also become available for patients with myelodysplastic syndrome, which eventually develops to aggressive AML. Their disease progression can be halted by drugs targeting DNA methylation and cytosine metabolism, namely 5-aza-cytidine (Vidaza) and 5-aza-2′-deoxycytidine (Dacogen). These chemically simple substances are, in spite of their limited effects on AML overall, presently the two most profitable AML drugs (Global Data, Pharma e-Track 2013). Sadly, for most AML patients, AML chemotherapy has not made significant progress in the last few years. It is still a 30 year old drug regime based on an anthracycline (Daunorubicin or Idarubicin) supplemented by arabinoside-C (ara-C) (A. Burnett, M. Wetzler, and B. Lowenberg, J Clin Oncol 29 (5), 487 (2011) and F. Ferrara and C. A. Schiffer, Lancet 381 (9865), 484 (2013)). Presently, complete remission is reached in 30-40% of AML patients less than 60 years old, and less than 10% in patients older than 70 years (Mehta 2010). However, relapse risk is in the range of 45-50% in older patients, making AML the leading cause of death due to leukemia with a 5-year relative survival below 20%. Intensive chemotherapy often is severe with lethal side-effects, such as lesions in hematopoietic tissue, particularly the bone marrow, as well as the intestine and the heart (Joel et al, A. Rohatiner, in Leukemia, edited by E. D. Henderson, T. A. Lister, and M. F. Greaves (Saunders, Philadelphia, 2002), pp. 394). There is thus a need for novel compounds that selectively target leukemia blasts, and leave normal tissues and cells largely unaffected. Drugs based on disease-related molecular alterations in AML cells have so far been disappointing. An example is patients whose AML cells constitutively express active Flt-3 tyrosine kinase, who benefit little from Flt-3 inhibitors. Thus, there is a major clinical need for new drugs in leukaemia therapy.
Iodinin (Scheme 1) has been known for almost a centennial, and is a member of the phenazine family. It is a planar nitrogen-containing heterocyclic compound produced by a variety of bacteria. Iodinin is an oxidized phenazine, namely 1,6-dihydroxyphenazine-5,10-dioxide, see Scheme 1.

Iodinin can be obtained in different ways, the first and the most widespread is by bacterial production. The first iodinin-producing bacterium, a terrestrial bacteria, Chromobacterium iodinum was named owing to the purple, bronze-glinting pigment which covers its colonies on suitable solid media (McIlwain H, Biochem J 1943). This pigment, the iodinin, was found to inhibit the growth of certain other bacteria. Iodinin is also produced by Pseudomonas phenazinium (Byng G S et al, J. Gen. Microbiol. 1976; 97: 57-62), when grown on a variety of carbon sources, especially L-threonine. A second carbon growth dependant is the biosynthesis by Brevibacterium iodinum (Gerber N N et al, Biochem 1967; 6(9): 2701-2705). The highest yield of iodinin production occurred in experiments with resting cells in the presence of some three-, four-, or five-carbon amino acids. Tricarboxylic acid cycle compounds, especially succinic acid, also gave high yields. Another biosynthesis by Arthrobacter paraffineus KY 7134 (Suzuki T et al, Agr. Biol. Chem. 1971; 35(1): 92-98), on n-paraffin as the only source of carbon, produced two sorts of crystalline pigments in the culture medium, one yellow and one deep-red, corresponding respectively to 1,6-dihydroxyphenazine (iodinin intermediate) and 1,6-dihydroxyphenazine-5,10-di-N-oxide (iodinin). Microorganisms forming a novel group of Nocardiaceae were seen to produce slants with lustrous coppery needles on the mycelium and in the agar, characteristic of Iodinin crystals (Gerber N N., 1966; 5(12): 3824-3829). Another production of iodinin, as a culture metabolite, is also possible after growth of Acidithiobacillus ferrooxidans on elemental sulfur (Ceskova P et al. Folia Microbial. 2002; 47(1): 78-80).
The second process for obtaining iodinin is through fungal production. Iodinin was isolated from a soil sample, Nocardiopsis dassonvillei (N. syringae, N. mutabilis and N. atra), an alkalophilic actinomycete, strain OPC-15, that produced different phenazine antibiotics under different culture conditions, including Iodinin. (Tsujibo H et al. Agric. Biol. Chem. 1982; 52(2): 301-306). Other Actinomycetes, e.g. microbispora amethystogenes and parva, streptosporangium album and amethystogenes, realize extracellular production of characteristic iodinin violet crystal (pigment) in oat-meal agar medium (Tanabe I et al. J. Ferment. Bioeng. 1995; 79 (4): 384-386). An efficient method could be fungal metabolite screening, showing the production of many mycotoxins and fungal metabolites, possibly containing iodinin (Nielsen K F. Journal of Chromatography A 2003; 1002: 111-136).
Iodinin is chemically related to the compound tirapazamine (SR-4233) which is an experimental anticancer drug. Tirapazamine also has the N-oxide functionality (Scheme 1) and is activated to its toxic form preferentially in the hypoxic areas of solid tumors. Thus the combination of tirapazamine with conventional anticancer treatments is particularly effective. Tirapazamine has undergone phase III testing in patients with head and neck cancer and gynecological cancer, and similar trials have been undertaken for other solid tumor types (Denny, W A “Prospects for hypoxia-activated anticancer drugs” Current Medicinal Chemistry 4 (5): 395-9, 2004).
Iodinin is also found as bioactive metabolites from marine biological resources. As the bio prospecting for marine compounds is expanding, it has been discovered that some Marine actinomycetes bacterium and marine Actinomadura sp. are proven to be the best, offering a great biological diversity and therefore a great chemical diversity. Other microorganisms like microbispora aerata, pseudomonas iodina, and streptomyces thioluteus are capable of synthesizing 1,6-Phenazinediol-5-oxide, an intermediate of the Iodinin biosynthesis (Gerber N N et al. Biochem 1965; 4 (1): 176-180).
The biological properties of the phenazine class of natural products include antibiotic, antitumor, antimalaria, and antiparasitic activities. The physiological function leading to these activities can be inhibition/control of DNA, RNA, and protein synthesis as well as disruption of energy requiring membrane-associated metabolic processes. The planar, aromatic iodinin core has structural similarities to known intercalators, e.g. daunorubicin, and thus acts as a DNA intercalating agent, with a much lower cardiac toxicity. The interaction phenazine-DNA was shown by differences in the comparison between the UV/visible spectrum of a phenazine in the presence of GC and AT-rich double-stranded oligonucleotides, and the spectrum of pure phenazine. Although no binding to single-stranded DNA was observed, the binding with double-stranded DNA occurred with strong association constants, in the 10−4-10−6 M−1 range, comparable to those of ethidium bromide (Hollstein U et al. Biochem 1971; 10 (3): 497-504). The use of iodinin and myxin is only briefly reported in the prior art. One study reported low activity against a mouse sarcoma model (Endo et al, Tohoku University. Ser. C, Medicine 14 (3), 169 (1967). Iodinin has a number of biological effects. In U.S. Pat. No. 3,764,679 iodinin is claimed to have antihypertensive effects.
It was recently reported that iodinin, extracted for a bio prospecting screen of marine actinomycetes bacteria, had a pronounced effect on AML cells (Myhren et al, Marine drugs 11 (2), 332 (2013) and was found to be particularly potent against leukaemia cell lines and AML-patient blasts. It was less toxic than DNR (at comparable anti-AML activity) towards peripheral blood leukocytes (PBL), rat cardiomyoblasts and blood platelets. Even direct infusion of a supra-saturated solution of iodinin into the gut of the mouse through a tube failed to cause any intestinal symptoms or histologically detectable alteration of the intestinal mucosa, like mucositis, which is common after anthracycline treatment. Thus, iodinin is an attractive potential drug for treatment of AML.
However, synthesis of phenazines and its derivatives are sparsely described in the prior art. One example is the Wohl-Aue reaction (Patcher I J et al. J. Am. Chem. Soc. 1951; 73 (10), 4958-4961), an organic reaction between an aromatic nitro compound and an aniline to form a phenazine in the presence of an alkali base. This method of synthesis was the first to have been used to synthesize the phenazine core. It has nonetheless a major issue; the reaction's yield is below 20%. Synthesis of phenazine derivatives by Cu-catalyzed homocoupling of 2-halogenoanilines in water has been proposed (Yu L et al. J Organomet Chem 2012; 705: 75-78)—see Scheme 2 below:

In spite of their promising biological effects, iodinin/myxin have several major disadvantages: a) they are practically insoluble in water, hindering in vivo testing in mammals; b) they can only be obtained by bio prospecting, which is laborious and expensive yielding only milligram quantities of the substance at a high cost and the process is time-consuming; no synthetic preparation process is known in the prior art for these two compounds; and c) they have a non-selective bio distribution in vivo and lack the chemical functionality required to attach functional groups for regulating their bio distribution. In the prior art, these problems are not properly addressed. In US 2009/042894 a biotechnological procedure is suggested to produce iodinin. In DE 2016467, DE 2115660, U.S. Pat. No. 3,929,790, U.S. Pat. No. 3,937,707, WO 2008/089283, and in Alonso et al, Chem. Comm. 2004, 41, 412-413, methods to alkylate iodinin or myxin via their alkali salts is described. However, the functional groups introduced were alkyl groups, rendering the derivatives less water soluble than the parent compounds. No discussion of improvement in water solubility was described.